CN1597981A - Process of extracting DAN from tomato ketchup for testing transgene component - Google Patents
Process of extracting DAN from tomato ketchup for testing transgene component Download PDFInfo
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- CN1597981A CN1597981A CN 200410020100 CN200410020100A CN1597981A CN 1597981 A CN1597981 A CN 1597981A CN 200410020100 CN200410020100 CN 200410020100 CN 200410020100 A CN200410020100 A CN 200410020100A CN 1597981 A CN1597981 A CN 1597981A
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Abstract
The invention relates to distill the DNA from the ketchup, which is used in detecting the components of the gene. The method has following features: adding the pretreatment to the procedure of distilling the DNA to make the pH value of the ketchup meet the requirements of the subsequent distilment; increasing the times of distilling the chloroform + isoamyl alcohol and the time of depositing the bromohexadecane trimethylamine to get rid of the protein, polysaccharide and other impurities; resolving the DNA solution into the 30mul deionized water, getting 15mul for once PCR reaction to increase the density of the DNA cyclostyle in the reaction system. The advantages are high speed, convenience, strong specificity and high.
Description
Technical field
Present method relates to the detection technique of transgene component in the deep processing food, especially extracts the method for the template DNA that is used for the transgene component detection from tomato-sauce.
Background technology
Fast development along with genetic engineering technique, a large amount of is that the genetically modified food that processes raw material enters the commercialization production and selling with genetically modified crops, add up according to internal authority mechanism, kind surplus the whole world reaches 4000 by the genetically modified food of genetically modified crops processing and food ingredient (referring to: U.S.Food and Drug Administration, Office of Food Additive Safety.Listof Completed Consultations on Bioengineering Food[DB/OL] .http: //www.fda-cfsan/biotechnology.2002-10.).At present still disputable to the security of genetically modified food.
The potential risks of genetically modified food cause that countries in the world government greatly pays close attention to, and therefore launch respectively the tag control system and the entry and exit product reporting system of genetically modified food.The requirement of United Nations and European Union is more strict, the regulation genetically modified food all needs to detect monitoring from studying, produce, transport, store, be sold to all links such as import and export, therefore an important process that becomes each main trading country check is identified in the detection of transgenic product, and the relevant stdn detection architecture of foundation, (referring to Zhu Guangfu, countries in the world are to the attitude and the management of genetically modified food, oil and foodstuffs science and technology to be adapted to various methods for detecting transgenic foods, 2001,3:1-5).
Use the transgene component in the PCR method detection deep-processed food, the ultimate challenge that is faced is the DNA composition that how to extract from numerous highly processed goods effectively as the PCR reaction template, the difficulty of its DNA extraction is that not only DNA is seriously damaged in the course of processing, and the factors such as the physics that produces in the course of processing, chemistry or biology also can have influence on the DNA quality.All kinds of different finely processed product that different work programs produces, the residual volume of DNA differs widely in the palliating degradation degree of its DNA and the product, therefore exploitation is the key that the deep-processed food transgene component detects at the template DNA extractive technique that the variant production transgenosis detects.
Summary of the invention
Present method is intended to set up from the tomato-sauce that processes with transgenic Fructus Lycopersici esculenti extracts template DNA, and the PCR that is used for transgene component detects.
The objective of the invention is to overcome deficiency of the prior art, a kind of method of extracting the DNA that is used for the transgene component detection from tomato-sauce is provided, it can be quick, easy, reliably, from tomato-sauce class deep-processed food, template DNA is extracted expeditiously, the qualitative detection that is used for transgene component is with the reliability of guaranteeing to detect.
Present method is at the characteristics of the pH value lower (2.0-3.0) of tomato-sauce, before formally carrying out DNA extraction, the pH value of the tomato-sauce that precipitates must be brought up to nearly neutrality.For this reason in adding the The pretreatment process, utilize 0.1mol/L Tris (pH8.0) buffered soln repetitive scrubbing tomato-sauce precipitation, the sedimentary pH value of tomato-sauce is brought up to more than 7.0, like this when adding bromohexadecane base Trimethylamine 99 extracting solution, the pH value of whole extraction buffer system is remained on about 8.0, and this is that DNA extraction is necessary.
Technical scheme of the present invention is summarized as follows:
Extract the method for the DNA that is used for the transgene component detection from tomato-sauce, it is made up of following step:
1. tomato-sauce is centrifugal, and precipitation is got 2-4g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 1-3 times of tomato-sauce volume 3-5 time.
2. the precipitation 2-4g that step 1 is obtained adds 10-15mL and extracts damping fluid through the bromohexadecane base Trimethylamine 99 of 65 ℃ of preheatings, mixing, 65 ℃ water-bath 1-1.5 hour, during put upside down mixing gently once every 10 minutes.
3. taking-up is left standstill and is cooled to 20-25 ℃, in 12000 rev/mins, centrifugal 12 minutes, gets supernatant.
4. adding and the isopyknic volume ratio of the resulting supernatant liquor of step 3 are 24: 1 trichloromethane: primary isoamyl alcohol, and slowly put upside down and shake up 5 minutes, 10000 rev/mins, centrifugal 10 minutes, get supernatant, add isopyknic volume ratio again and be 24: 1 trichloromethane: the primary isoamyl alcohol extracting is once got supernatant.
5. add the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes, shake up, 20-25 ℃ left standstill 50-90 minute, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
6. precipitation adds 1.2mol/L NaCl solution 1mL, dissolution precipitation, and 20-25 ℃ left standstill 5 minutes, added isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, put upside down and shake up 1 minute, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
7. the 3mol/L sodium acetate and the isopyknic 4 ℃ of pre-cold isopropanols that add the institute's supernatant liquor of getting 1/10 volume, slow mixing, 4 ℃ of standing over night, centrifugal 15 minutes, are abandoned supernatant by 14000 rev/mins.
8. be 70% washing with alcohol precipitation with 4 ℃ of precoolings, 400 μ L volume percent, remove residual ethanol, drying precipitated, obtain the DNA precipitation.
9. add the deionized water of 25-35 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
The invention has the beneficial effects as follows: the sedimentary pH value of tomato-sauce is brought up to more than 7.0, satisfied the requirement that transgene component detects, this method is quick, easy, high specificity, the efficient height, and the PCR that is fit to transgene component in the tomato-sauce based article detects.
Description of drawings
Endogenous special pcr amplification result (the 1.DNA Marker of Fig. 1 tomato-sauce DNA with reference to the PG34 gene; 2. bright tomato; 3. tomato seeds; 4. tomato-sauce)
Embodiment
1. material and reagent
1) material: the tomato-sauce sample is available from the supermarket, contrasts to be bright tomato and tomato seeds.
2) extract reagent:
DNA extraction damping fluid (1L): dissolving bromohexadecane base Trimethylamine 99 20g in the 1000ml water, Tris 12.114g, NaCl 81.816g, Na
2EDTA 7.444g, the PVP[polyvinylpyrrolidone] 20g, autoclaving.
Bromohexadecane base Trimethylamine 99 precipitated liquid (1L): dissolving bromohexadecane base Trimethylamine 99 5g in the 1000ml water, NaCl 2.34g, autoclaving.
Sodium chloride solution (1.2mol/L): dissolving NaCl 70g in the 1000ml water, autoclaving.
Sodium acetate solution (3mol/L): dissolving 408.1g sodium acetate trihydrate in the 800ml water, transfer pH to 5.2 with glacial acetic acid, add water and be settled to 1L, autoclaving.
Tris-HCl solution (0.1mol/L, pH8.0): with 800ml water dissolution 12.114g Tris, be cooled to room temperature, transfer pH to 8.0, be settled to 1L with concentrated hydrochloric acid.
TE damping fluid: Tris 0.01mol/L, Na
2EDTA 1.0mmol/L transfers pH to 8.0, autoclaving with hydrochloric acid.
Trichloromethane: primary isoamyl alcohol (24: 1); 70% ethanol; Virahol; The deionization sterilized water.
2.DNA the flow process of extracting:
1) sample pretreatment
Get liquid processed sample 30mL such as tomato-sauce, room temperature, 10000 rev/mins, centrifugal 15 minutes, abandon supernatant, get precipitation 2-4g and change in the mortar of precooling, place on ice and grind, all change in the 50mL centrifuge tube, use 0.1mol/LTris-HCl solution repetitive scrubbing sample 3-5 time, method is for adding 30mL 0.1mol/L Tris-HCl solution, shake up repeatedly, in 4 ℃, 10000 rev/mins, abandoned supernatant in centrifugal 15 minutes, sedimentary pH value is reached near neutral.This precipitation is used for DNA extraction.
2) learn from else's experience pretreated sample adds 10mL and extracts damping fluid, mixing through the bromohexadecane base Trimethylamine 99 of 65 ℃ of preheatings.65 ℃ of water-baths 1 hour, during put upside down mixing gently once every 10 minutes.
3) leave standstill and be cooled to room temperature, 12000 rev/mins, centrifugal 12 minutes, get supernatant.
4) add isopyknic trichloromethane+primary isoamyl alcohol and slowly put upside down and shake up 5 minutes, in 10000 rev/mins, centrifugal 10 minutes, get supernatant, add isopyknic trichloromethane+primary isoamyl alcohol and repeat extracting once, get supernatant.
5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and room temperature left standstill 1 hour, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
6) precipitation adds 1.2mol/L NaCl solution 1mL dissolution precipitation, and room temperature left standstill 5 minutes, and adding equal-volume trichloromethane+primary isoamyl alcohol is put upside down and shaken up 1 minute, in 12000 rev/mins, centrifugal 10 minutes, gets supernatant.
7) 3mol/L sodium acetate and isopyknic cold isopropanol of adding 1/10 volume, slow mixing, 4 ℃ of standing over night, centrifugal 15 minutes, are abandoned supernatant by 14000 rev/mins.
8) add 400 μ L, 70% cold washing with alcohol precipitation, remove residual ethanol, drying precipitated.
9) add the DNA precipitation of extracting in the deionized water dissolving tomato-sauce of 30 μ L sterilization, get the template of 15 μ L as the PCR reaction; DNA extraction thing with bright tomato that compares or tomato seeds dissolves with 100 μ L TE, and it is standby to put-20 ℃ of preservations.
10) whether extract the DNA that can be used for the PCR detection, adopt the qualitative TRAP of special endogenous reference gene PCR to identify.Experimental technique and condition are as follows:
(1) the PCR primer sequence of special endogenous reference gene (seeing Table 1)
The PCR primer sequence of the special endogenous reference gene of table 1
Detect gene primer sequence amplification fragment length raw material crop
Just: 5 '-gga tcc tta gaa gca tct agt-3 '
PG34 383 bp tomatoes
Instead: 5 '-cgt tgg tgc atc cct gca tgg-3 '
(2) the PCR reaction system of special endogenous reference gene is formed (seeing Table 2)
The amount of each reagent is suitably adjusted according to the cumulative volume of reaction system in the reaction system, and each reaction system should be provided with two parallel pipes.The reaction system that tomato-sauce DNA quality examination is adopted is identical.
Table 2 PCR detects reference reaction system (25 μ L system)
Reagent name adds the amount of PCR reaction system
10 * PCR reaction buffer (contains Mg
2+) 2.5 μ L
DNTP solution (every kind is 10mmol/L) 0.5 μ L
Forward primer (10pmol/ μ L) 2.5 μ L
Reverse primer (10pmol/ μ L) 2.5 μ L
Taq enzyme (2U/ μ L) 2.0 μ L
*Template DNA 10～15 μ L
Water is supplied reaction system, and making cumulative volume is 25 μ L
*: the dna profiling consumption of positive control sample is 1 μ L in the table.
(3) the PCR reaction conditions of special endogenous reference gene (seeing Table 3)
The PCR of the special endogenous reference gene of table 3 detects the reference reaction condition
The gene sex change amplification cycles number of times that is amplified extends at last
94℃，30s
PG34 94℃，3min 60℃，30s 35 72℃，5min
72℃，45s
(4) deposition condition
Sepharose (gel thawing back room temperature adds ethidium bromide when being placed into 60 ℃ of left and right sides, and content is 0.5g/mL) with 1 * TAE electrophoretic buffer preparation 2%.With PCR product and the sample-loading buffer uniform mixing of 10 μ L, join then in the gel pore in proportion, (3V/cm～5V/cm) carry out electrophoresis, electrophoresis time is 40min～60min to select suitable voltage.Place sepharose on the gel imaging instrument or observation and photograph on the ultraviolet transilluminator.
Below in conjunction with specific embodiment the present invention is further described.
Embodiment 1:
Jinsui River board tomato-sauce of producing with Tianjin Tong Mao industry and trade company limited is material, adopts present method to extract tomato-sauce DNA,
(1) tomato-sauce is centrifugal, precipitation is got 3g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 1 times of tomato-sauce volume 3 times, makes the approaching neutrality of sedimentary pH value.
(2) the precipitation 3g that step (1) is obtained adds 10-15mL and extracts damping fluid through the bromohexadecane base Trimethylamine 99 of 65 ℃ of preheatings, mixing, 65 ℃ water-bath 1-1.5 hour, during put upside down mixing gently once every 10 minutes.
(3) taking-up is left standstill and is cooled to 20-25 ℃, in 12000 rev/mins, centrifugal 12 minutes, gets supernatant.
(4) adding isopyknic volume ratio is 24: 1 trichloromethane: primary isoamyl alcohol, and slowly put upside down and shake up 5 minutes, 10000 rev/mins, centrifugal 10 minutes, get supernatant, add isopyknic volume ratio again and be 24: 1 trichloromethane: the primary isoamyl alcohol extracting is once got supernatant.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 60 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6) precipitation adds 1.2mol/L NaCl solution 1mL dissolution precipitation, and 20-25 ℃ left standstill 5 minutes, adds isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, and put upside down and shake up 1 minute, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
(7) 3mol/L sodium acetate and isopyknic 4 ℃ of pre-cold isopropanols of adding 10%, slow mixing, 4 ℃ of standing over night, centrifugal 15 minutes, are abandoned supernatant by 14000 rev/mins.
(8) with 4 ℃ of precoolings of 400 μ L, volume percent is 70% washing with alcohol precipitation, removes residual ethanol, and is drying precipitated, obtains the DNA precipitation.
(9) add the deionized water of 30 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
(1) tomato-sauce is centrifugal, precipitation is got 2g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 1 times of tomato-sauce volume 5 times.
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 90 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 25 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
(1) tomato-sauce is centrifugal, precipitation is got 2g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 1 times of tomato-sauce volume 5 times.
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 90 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 25 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
(1) tomato-sauce is centrifugal, precipitation is got 2g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 3 times of tomato-sauce volumes 5 times,
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 70 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 25 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
Embodiment 5
(1) tomato-sauce is centrifugal, precipitation is got 4g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 1 times of tomato-sauce volume 3 times.
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 60 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant;
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 25 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
Embodiment 6
(1) tomato-sauce is centrifugal, precipitation is got 3g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 2 times of tomato-sauce volumes 5 times,
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 75 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 35 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
Embodiment 7
(1) tomato-sauce is centrifugal, precipitation is got 3g precipitation, with the solution washing of the 0.1mol/LTris-HCl of the pH=8.0 of 2 times of tomato-sauce volumes 3 times.
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 80 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 35 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
Embodiment 8
(1) tomato-sauce is centrifugal, precipitation is got 2.5g precipitation, with the 0.1mol/LTris-HCl solution washing of the pH=8.0 of 1 times of tomato-sauce volume 4 times.
(2)-(4) step is with embodiment 1.
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 90 minutes, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant.
(6)-(8) step is with embodiment 1.
(9) add the deionized water of 25 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
Claims (6)
1. extract the method for the DNA that is used for the transgene component detection from tomato-sauce, it is made up of following step:
(1) tomato-sauce is centrifugal, precipitation is got 2-4g precipitation, with the solution washing of the 0.1mol/L Tris-HCl of the pH=8.0 of 1-3 times of tomato-sauce volume 3-5 time;
(2) the precipitation 2-4g that step (1) is obtained adds 10-15mL and extracts damping fluid through the bromohexadecane base Trimethylamine 99 of 65 ℃ of preheatings, mixing, 65 ℃ water-bath 1-1.5 hour, during put upside down mixing gently once every 10 minutes;
(3) taking-up is left standstill and is cooled to 20-25 ℃, in 12000 rev/mins, centrifugal 12 minutes, gets supernatant;
(4) adding and the isopyknic volume ratio of the resulting supernatant liquor of step (3) are 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 10000 rev/mins, centrifugal 10 minutes, get supernatant, add isopyknic volume ratio again and be 24: 1 trichloromethane: the primary isoamyl alcohol extracting is once got supernatant;
(5) the bromohexadecane base Trimethylamine 99 precipitated liquid of 2 times of volumes of adding shakes up, and 20-25 ℃ left standstill 50-90 minute, 12000 rev/mins, centrifugal 10 minutes, abandoned supernatant;
(6) precipitation adds 1.2mol/LNaCl solution 1mL, dissolution precipitation, and 20-25 ℃ left standstill 5 minutes, added isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, put upside down and shake up 1 minute, 12000 rev/mins, centrifugal 10 minutes, get supernatant;
(7) add 3mol/L sodium acetate and isopyknic 4 ℃ of pre-cold isopropanols of the institute's supernatant liquor of getting 1/10 volume, slow mixing, 4 ℃ of standing over night, centrifugal 15 minutes, are abandoned supernatant by 14000 rev/mins;
(8) be 70% washing with alcohol precipitation with 4 ℃ of precoolings, 400 μ L volume percent, remove residual ethanol, drying precipitated, obtain the DNA precipitation;
(9) add the deionized water of 25-35 μ L, the DNA precipitation that dissolving is extracted from tomato-sauce through sterilization.
2. the method for extracting the DNA that is used for the transgene component detection from tomato-sauce according to claim 1, it is characterized in that: Tris-HCl solution is 1 times of tomato-sauce volume in the described step (1).
3. the method for extracting the DNA that is used for the transgene component detection from tomato-sauce according to claim 1, it is characterized in that: the number of times with the Tris-HCl solution washing in the described step (1) is 3 times.
4. the method for extracting the DNA that is used for the transgene component detection from tomato-sauce according to claim 1, it is characterized in that: the resulting sedimentary pH value of described step (1) is 7.0.
5. the method for extracting the DNA that is used for the transgene component detection from tomato-sauce according to claim 1, it is characterized in that: the time of repose in the described step (5) is 60 minutes.
6. the method for extracting the DNA that is used for the transgene component detection from tomato-sauce according to claim 1 is characterized in that: the volume of sterilization deionized water is 30 μ L in the described step (9).
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| CN107365767A (en) * | 2017-09-15 | 2017-11-21 | 广东美格基因科技有限公司 | A kind of method that DNA is extracted from soy sauce |
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| CN107365767A (en) * | 2017-09-15 | 2017-11-21 | 广东美格基因科技有限公司 | A kind of method that DNA is extracted from soy sauce |
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